The Prostate 72:1789^1801 (2012)

Down-Regulation of Calcium/Calmodulin-Dependent
Protein Kinase Kinase 2 by Androgen Deprivation Induces
Castration-Resistant Prostate Cancer
Takashi Shima,1 Atsushi Mizokami,1* Toru Miyagi,1 Keiichi Kawai,2 Kouji Izumi,1Misako Kumaki,1 Mitsuo Ofude,1 Jian Zhang,3 Evan T. Keller,4 and Mikio Namiki1
1

Department of Integrative CancerTherapyand Urology, Kanazawa University Graduate Schoolof Medical Sciences,
Ishikawa, Japan
2
Division of Health Sciences,Graduate Schoolof Medical Science, Kanazawa University, Ishikawa, Japan
3
Guangxi Medical University, Pharmacologyand Biomedical Sciences,Guangxi,China
4
Departments of Urologyand Pathology,University of Michigan, Ann Arbor, Minnesota

BACKGROUND. Conversion into androgen-hypersensitive state and adaptation to the low
concentration of androgen during ADT cause relapse of prostate cancer (PCa). It is important
to identify differentially expressed genes between PCa and normal prostate tissues and toreveal the function of these genes that are involved in progression of PCa.
METHODS. We performed cDNA microarray analysis to identify differentially expressed
genes, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2). Immunohistochemical analysis was conducted to investigate the relationship between the CAMKK2 expression level and prognosis. The function of CAMKK2 was assessed by generating
CAMKK2 overexpressed LNCaP cells and by knockdown of CAMKK2.
RESULTS. We identified CAMKK2 overexpressed six times in PCa more than normal prostate by cDNA microarray analysis. Immunohistochemical analysis of CAMKK2 protein
showed that CAMKK2 protein was expressed more in PCa than normal tissue. However,
the expression in the high-grade PCa diminished. Moreover, the narrowness of CAMKK2positive area before ADT was a poor prognostic factor. Androgen-deprivation treatment
from the medium in which LNCaP cells were cultured in the presence of 10 nM DHT repressed CAMKK2 expression. CAMKK2 overexpressed LNCaP cells (LNCaP/GFPCAMKK2) attenuated androgen-sensitivity. Tumorigenesis of LNCaP/GFP-CAMKK2
cells in male SCID mice was decreased compared with control cells irrespective of castration.
Finally, knockdown of CAMKK2 mRNA in LNCaP cells induced androgen-hypersensitivity
and stimulated LNCaP cell proliferation.
CONCLUSIONS. Induction of androgen-hypersensitivity after ADT may be involved in
down-regulation of CAMKK2. This result may provide new therapeutic approach to
keep androgen-sensitivity of PCa after ADT. Prostate 72: 1789–1801, 2012. # 2012 Wiley
Periodicals, Inc.

KEY WORDS:CAMKK2; prostate cancer; androgen-hypersensitivity; AMPK

Additional supporting information may be found in the online
version of this article.
Grant sponsor: Ministry of Education, Culture, Sport, Science, andTechnology of Japan; Grant number: 21592037.
The authors declare no conflict of interest.
*Correspondence to: Atsushi Mizokami, Department of Integrative
Cancer Therapy and Urology, Kanazawa University Graduateß 2012 Wiley Periodicals,Inc.

School of Medical Sciences, 13-1 Takaramachi, Kanazawa, Ishikawa
920-8640, Japan. E-mail: mizokami@med.kanazawa-u.ac.jp
Received 29 January 2012; Accepted 3 April 2012
DOI 10.1002/pros.22533
Published online 1 May 2012 in Wiley Online Library(wileyonlinelibrary.com).

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INTRODUCTION

Prostate cancer (PCa) is the most common malignancy and the second leading cause of cancer-related
death of men in the United States [1]. Since advanced
PCa is initially dependent upon androgens, androgen-deprivation therapy (ADT) is the first choice foradvanced PCa. Improvement of symptom or decreasein prostate-specific antigen (PSA) is observed in
more than 90% of PCa patients by ADT. Unfortunately, after an initial response to ADT, PCa eventuallyloses responsiveness to the androgen blockade andprogresses into castration-resistant prostate cancer(CRPC).
Although multiple molecular mechanisms that
could explain the development to CRPC havebeen proposed [2], androgen receptor (AR) is
still a key mediator in the progression of PCa [3].
Especially, an androgen-hypersensitive situationof PCa plays an important role in an adaptation
to castrate levels of androgen that is secreted
from adrenal gland. Furthermore, intratumoral androgen synthesis in PCa or bone metastasis region
also supplies androgen enough for androgenhypersensitive PCa cells to survive and proliferate[4,5]. Moreover, not only cancer cells but also cancerassociated fibroblasts coordinately synthesize dihydrotestosterone (DHT) from adrenal androgen
DHEA, activate AR, and enhanced the cell proliferation after ADT [5].
As for androgen-hypersensitivity, CRPC is induced
by several pathways: (A) promiscuous AR mutations.
AR with T877A and H874Y mutation responds to various ligands, such as DHEA, estradiol, and progesterone [6,7]. (B) AR overexpression by AR gene
amplification [8], increase of AR promoter activity, orAR mutation that increase AR stability [9]. (C) Increase of AR coactivators. Expression AR coactivator
ARA55 and TIFII was increased in some of CRPC
patients [10,11]. (D) Outlaw pathways through transactivation of kinases, or crosstalk with cytokines or
growth factor receptors [2]. AR coactivator p300 andinterleukin-6 coordinately activate AR and induce
PSA [12].
To identify biomarkers for PCa and key proteinsthat induce carcinogenesis and progression during
ADT, gene expression profiling between normal prostate and PCa was investigated using cDNA microarray technique [13–16]. As a result, many molecules
have been identified as PCa markers, such as AMACR(a-methylacyl coenzyme A racemase), as prognostic
biomarkers, such as MUC1 and AZGP1 [17], and as
the proteins that induce progression such as the polycomb gene EZH2 [18]. Holzbeierlein et al. [13] demonstrated that overexpression of AR mRNA was also
The Prostate

observed in CRPC. Overexpression of the AR could
contribute to androgen-hypersensitivity in the low
concentration of androgen. Moreover, they also identified some of the genes that were differentiallyexpressed in resistant tumors and that could contribute to the process of reactivation.
In the present study, we also identified many
genes that were differentially expressed between
normal prostate and PCa tissue, and investigated
the functions of them. Then we focused on one ofoverexpressed genes in PCa, calcium/calmodulindependent protein kinase kinase 2 (CAMKK2). Recently, Frigo et al. [19] and Massie et al. [19] reported
that CAMKK2 induced by androgen in PCa cells
stimulated migration, invasion, and cell growth.
However, we report that the size of CAMKK2positive area was inversely correlated with prognosis
and that down-regulation of CAMKK2 expression by
ADT increases the AR function and induces androgen-hypersensitivity.
MATERIALS ANDMETHODS
cDNAMicroarray Analysis
In order to identify the differentially expressed
genes between normal prostate and PCa tissue,
we compared expression profile between each of
three normal prostate biopsy samples and eachof three PCa samples using cDNA microarray analysis. The characteristics of the patients were describedin Table I. cDNA microarray analysis was performed
in DNA Chip Research Inc. (Kanagawa, Japan) after
sending biopsy samples from the patients and analyzed by Whole Human Genome Microarray 4  44K(Agilent Technologies; GEO accession number:
GSE30994). Needle biopsies were performed after weobtained informed consent from patients with the
document approved from the Graduate School of
Medical Science, Kanazawa University.
Cell Lines and Generation of StableTransformant
LNCaP and VCaP cells (ATCC, Manassas, VA)
were cultured in DMEM supplemented with 1%
penicillin/streptomycin (Invitrogen, Carlsbad, CA)
and 5% and 10% fetal bovine serum (Sigma–Aldrich,
St. Louis, MO), respectively. To generate CAMKK2overexpressed LNCaP cells, LNCaP cells were transfected with 0.5 mg pCMV6-AC-GFP or pCMV6AC-GFP-CAMKK2 from OriGene Technologies(Rockville, MD) using Lipofectamine reagent (Invitrogen). Eight hours after transfection, the cells were cultured at 1,200 mg/ml G418 (Sigma–Aldrich) andselected as stable GFP and GFP-CAMKK2 overexpressing cells.

Repression of CAMKK2 Induces Androgen-Hypersensitivity
TABLE I. Characteristics of the Patients

Patients

Age

PSA(ng/ml)Gleason score

Stage

P01
P02
P03
P11
P12
P13

59
74
70
77
72
82

3.6
8.97
5.09
55
227.8
96.7

BPH (33 g)
BPH (67 g)
BPH (50 g)
4þ4¼8
4þ4¼8
4þ3¼7

T3a, N0, M0
T4, N1, M0
T3a, N0, M0

BPH, benign prostate hypertrophy.

Cell Proliferation Assay
Twenty-four hours after cells were plated at a density of 5  104 cells onto 12-well plates with MDEM5% charcoal-stripped fetal calf serum (CCS; Thermo
Scientific HyClone, UK), cells were treated with orwithout DHT in DMEM-5% CCS and the medium
was changed every 2 days. In each experiment, cells
were harvested and the numbers of the cells werecounted in triplicate using a hemocytometer. The data
represent the means  SD of three replicates.
RNAInterference Analysis
The specific Stealth CAMKK2 and AR small interfering RNAs (siRNAs) were synthesized by Invitrogen. CAMKK2 target siRNA-01 and -02 sequence
were 50 -UUCGAACACCAUGUACAGAUGGUCC-30 ,50 -CAAUGAAGGACUCCAUGCCCAGG-UG-30 , respectively. Non-target (NT) siRNA were purchasedfrom Invitrogen. For CAMKK2 knockdown, LNCaP
cells were plated into 12-well plates at 5  104 cells/
well. After cultured for 24 hr, cells were transfectedwith each density of 20 nM CAMKK2 siRNA or NT
siRNA using Lipofectamine RNAiMAX Reagent(Invitrogen) for 24 hr. Total RNA or total proteins
were exacted after siRNA transfection. For cell proliferation, after transfection of cells with NT siRNA or
CAMKK2 siRNA-01 for 12 hr, cells were treated with
0, 0.1, 1.0, and 10 nM DHT, and the numbers of thecells were counted 4 days after transfection. Medium
was changed every 2 days and DHT was also added
to the medium.
RT-PCR
Total RNA extraction from cell cultures and RTPCR was performed followed as described previously[5]. The sense and anti-sense primers used RT-PCR ofCAMKK2 are 50 -CTGGACATGAACGGACGCTGCATCT-30 and 50 -GCCCTTGGTTGACCAGTTCGA-ACA
C-30 , respectively. The annealing temperature of RT-

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PCR of CAMKK2 was 658C and PCR cycle number
was 30 cycles. The amplified PCR products were visualized using electrophoresis on a 1.5% agarose gel.
For quantification of mRNA expression, the gene expression in each sample was normalized by GAPDH
gene expression.
Luciferase Assay
To evaluate AR transcriptional activity, 24 hr after
plating 5  104 cells on 12-well plates in DMEM-5%
CCS, LNCaP and its sublimes were transfected using
Lipofectamine transfection reaction (Invitrogen) using
0.5 mg of luciferase reporter plasmid, pGLPSAp-5.8,
driven by a 5.8 kb PSA promoter including 3 androgen-response elements (ARE) [21]. Twelve hours after
transfection, cells were treated by the addition of indicated concentration of DHT for 24 hr. After treatedcells were harvested, cells were lysed in luciferaselysis buffer (Promega, WI). As for knockdown
CAMKK2 expression in LNCaP cells, 5  104 LNCaP
cells were transfected with 20 nM NT siRNA or
CAMKK2 siRNA (Invitrogen) using RNAiMAX (Invitrogen) for 12 hr and then LNCaP cells were furthertransfected with pGL3PSAp-5.8 for 12 hr. Afterchanging medium, transfected LNCaP cells were cultured for 24 hr and harvested.
Western Blot Analysis
After finishing various treatments, total protein was
extracted from cells as described previously [22].
Protein was quantified according to the method of
Bradford, and equal amounts of protein were electrophoresed on a 10% or 12.5% Ready Gel J (Bio-Rad,
Hercules, CA). Membranes were incubated with mousemonoclonal antibody against CAMKK2 (Sigma–
Aldrich), GAPDH (Novus Biologicals, Littleton, CO),AR (NH27) [23]. Horseradish peroxidase-conjugatedsecondary antibody against anti-mouse monoclonal oranti-rabbit monoclonal antibody was used and protein
bands were visualized and quantitated with chemiluminescent reagent (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL) and ChemiDoc
XRS (Bio-Rad)
Animal Study
Severe combined immunodeficient (SCID) mice
were castrated 1 week before implantation of
LNCaP/GFP and LNCaP/GFP-CAMKK2 cells. The
cells were combined with 50% Matrigel (Collaborative
Biomedical Products, Bedford, MA) and 2  106 cells
were prepared for injection into the dorsal subcutis of
6-week-old male SCID mice castrated 1 week ago and6-week-old male SCID mice simultaneously. The
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dorsal tumor volume and body weights were monitored. Tumor growth was assessed by electronic
caliper measurement of tumor diameter in two
dimensions, and the tumor volume (V) was calculated
from the length (L) and width (W) according to theformula V ¼ LW2/2. Serum from mice was taken for
enzyme immunoassay of prostate-specific antigen(PSA) just before sacrifice and was frozen until measurement. To measure the concentration of PSA in serum of SCID mice, we asked SRL (Special Reference
Laboratories, Inc. Japan). This animal protocol wasapproved by the Institutional Animal Care and Use
Committee of the Graduate School of Medical Science,
Kanazawa University.
Immunohistochemistry of CAMKK2TM0016 tissue microarrays (TMAs) comprised of
80 cores containing normal tissue, matched for Gleason score at Sutgery were purchased from Provitro(Berlin, Germany) and PR952 TMAs comprised of 95
cores were purchased from Biomax (Rockville, MD).
The procedure for immunohistochemical staining(IHC) was performed using a Dako ChemMate
ENVISION Kit/HRP(DAB)-universal kit (K5007)
according to the manufacturer’s protocol (Dako,
Carpinteria, CA). Tissue specimens were stained withmouse monoclonal antibodies against CAMKK2 (a dilution of 1:200; Sigma–Aldrich). Japanese patient’s
prostate tissues were obtained from needle biopsyof prostate on diagnosis in Kanazawa University
Hospital. We picked up advanced PCa patients withbone metastasis. We obtained informed consent ofpatients for research and were admitted our researchin the Institutional Review Board of the Graduate
School of Medical Science, Kanazawa University.
The procedure for IHC staining was the same of tissue microarray except dilution of the primary antibody at 1:400.
Immunofluorescence Staining
Methods of immunofluorescence staining was followed by previous study [24]. Briefly, staining for
CAMKK2 protein was performed by overnight incubation using commercial kits in accordance withthe manufacturer’s instructions (Phallotoxins andZenonTM Tricolor Mouse and Rabbit IgG Labeling
Kit; Molecular Probes, Eugene, OR). We compared
the rate of a regional area with positive CAMKK2 expression with PSA-progression-free survival (PFS).
Statistical Analysis
Statistical significance was determined by using
the Prism 4.0 software. The chi-square test was
The Prostateutilized to assess the significance between different
proportions. Analysis of continuous variables between different groups was assessed by one-way
analysis of variance followed by Fisher’s protected
least significant difference test. , , and  represent significant difference P < 0.05, P < 0.01, andP < 0.001, respectively. Kruskal–Wallis test was used
to determine the statistical significance of differences
in IHC staining of tissue microarray. Logistic regression analysis was used to determine correlation of
CAMKK2 staining area and PFS.
RESULTS
Identification of Differentially Expressed CAMKK2
Between Prostate Cancer and Normal Prostate
To identify differentially expressed genes between
normal prostate and PCa tissue, approximately 40,000
genes were screened by cDNA microarray analysis
using mRNA from six different prostate biopsy samples from as described in Materials and Methods section (Table I). Seventy-nine (0.2%) of screened genes
were up-regulated (2.5-fold) and 124 (0.31%) ofgenes were down-regulated in each of three PCa samples (0.25-fold) compared with each of three normal
prostate samples. Then we focused on CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2)
which was a relatively highly expressed and >6-foldup-regulated in three PCa tissues compared withthree normal prostate tissues.
First, we investigated if the CAMKK2 expression
pattern on cDNA microarray is correlated with clinical PCa samples. As shown in Figure 1A–C, we classified intensity of the CAMKK2 staining of cytoplasm
in three phases. Immunohistochemistry (IHC) of
CAMKK2 using tissue microarray revealed that 87.8%(115/131) cases of primary PCa tissue and 85.7% (6/7) cases of bone metastasis showed positive staining
in cytoplasm, whereas only 4.2% (1/24) of normal
prostate showed positive staining (P < 0.01; Fig. 1D).
Interestingly, the ratio of CAMKK2-negative staining
rather diminished in the PCa tissue with the highGleason score 8, 9, and 10 significantly comparedwith low Gleason score (P ¼ 0.014). Therefore, we
also investigated the correlation between CAMKK2
expression and prognosis (progression-free survival,
PFS) of Japanese advanced PCa patients treated with
first-line androgen-deprivation therapy (ADT). All
patients had a diagnosis of the advanced PCa withbone metastases. Since PCa is multifocal disease andthe transected tissue area can vary significantly incomposition of cell type, we evaluated biopsy samples at the area of CAMKK2-positive cells. As shownin Figure 1E, regardless of an intensity of the expression of CAMKK2, the patients with area that

Repression of CAMKK2 Induces Androgen-Hypersensitivity

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Fig. 1. Immunohistochemistry of CAMKK2 in prostate tissue.Representative examples of photomicrographs (100  magnification) showing CAMKK2 expressionin the normalprostate andprostate cancer on tissuemicroarray analysis.A: CAMKK2 expressionin normalprostate
tissue (intensity). B: CAMKK2 expression in prostate cancer with Gleason score 8 (intensityþ).C: CAMKK2 expression in prostate cancer
with Gleason score 7 (intensityþþ). D: Immunohistochemistry of CAMKK2 on prostate tissue microarray. E: Kaplan^Meier estimates of
PSA-progression free survival for the indicated CAMKK2-positive area. The patients were classified in two groups by the rate of theCAMKK2-positive area (solid line: the rate of CAMKK2-positive area is more than 50%, dash line: the rate of CAMKK2-positive area is<50%).

CAMKK2 expression positive cells occupied <50%
had a poorer PFS significantly than the patientswith the area that CAMKK2 expression positive cells
occupied was bigger than 50% (median PFS 6 vs.
19 months, respectively; P ¼ 0.0031).
Androgen Induces CAMKK2 Expression
Since PCa is generally androgen-dependent before
ADT, we confirmed if dihydrotestosterone (DHT)
can induce CAMKK2 in LNCaP cells. DHT-induced
CAMKK2 mRNA in a dose-dependent manner in
24 hr (Fig. 2A). Western blot analysis also revealed
that DHT-induced 72 kDa CAMKK2 protein expression in a dose-dependent manner. In contrast, androgen-deprivation treatment from the medium in which
LNCaP cells were cultured in the presence of 10 nM

DHT gradually repressed CAMKK2 mRNA and protein expression (Fig. 2B).
Overexpression of CAMKK2 in LNCaP Cells
Represses Cell Proliferation
To explore the function of CAMKK2 on PCa cells,
we first generated stably CAMKK2 overexpressed
LNCaP cells (LNCaP/GFP-CAMKK2). Ninety-nine
kDa GFP-fused CAMKK2 protein was dominantly
expressed in cytoplasm of LNCaP/GFP-CAMKK2
cells (Fig. 2B and C). DHT of various concentrations
did not change AR mRNA expression in both
LNCaP/GFP and LNCaP/GFP-CAMKK2 cells. In
contrast, AR protein level in LNCaP/GFP-CAMKK2
cells was lower than that in LNCaP/GFP cells irrespective of DHT (Fig. 2D).
The ProstateFig. 2. Induction of CAMKK2 by androgen andgeneration of stable transformant of CAMKK2 in LNCaP cells. A: LNCaP cells were treatedwith DHT (0, 0.1, 1.0, and 10 nM) for 24 hr and total RNAs were extracted and subjected to real-time RT-PCR of CAMKK2 and GAPDH.
Whole cell lysate from each sample was also subjected to Western blot analysis. Anti-CAMKK2 antibody and anti-GAPDH antibody wereused for detection of 72 kDa CAMKK2 and 37 kDa GAPDH protein, respectively. B: Twenty-four hours after seeding of LNCaP cells,
cells were treated with 10 nM DHT for12 hr.Then the medium was changed to DMEM-5% CCS to remove DHTand the cells were cultured
for 12, 24, and 36 hr and harvested.The expression level of CAMKK2 mRNA and protein was subjected to real-time RT-PCR and Western
blot analysis. C: Generation of stable transformant of GFP or GFP-CAMKK2 in LNCaP cells. Total RNA and whole cell lysate from
LNCaP/GFP and LNCaP/GFP-CAMKK2 were subjected to RT-PCR and Western blot analysis, respectively. D: Immunofluorescenceanalysis of CAMKK2 in LNCaP/GFP and LNCaP/GFP-CAMKK2. Immunofluorescence analysis were performed using mouse anti-CAMKK2
antibody (red) as described in the Materials and Methods Section, and the blue signal represents nuclear DNA staining (400 magnification).
E: After cells were treated with 0, 0.1, 1.0, and 10 nM DHT for 24 hr, total RNAs were extracted and subjected to RT-PCR of AR and
GAPDH.Whole celllysates were subjected toWesternblot analysis of110 kDa AR and GAPDH 48 hr after10 nMDHT-treatment.

The ProstateRepression of CAMKK2 Induces Androgen-Hypersensitivity
We further performed luciferase reporter assay toconfirm how CAMKK2 overexpression affect AR
activity in LNCaP cells. LNCaP/GFP and LNCaP/
GFP-CAMKK2 cells were transfected with a luciferase
reporter (pGL3PSAp-5.8) driven by 5.8 kbp PSA promoter that includes strong 4.1 kbp upstream androgen-response element (ARE3) and weak ARE1 and 2[25]. The PSA promoter activity induced by DHT in
LNCaP/GFP-CAMKK2 cells was two to four times
lower than in LNCaP/GFP cells (Fig. 3A). Moreover,1795

basal level of PSA promoter activity in the absence of
DHT was also reduced in LNCaP/GFP-CAMKK2
cells by 2.7-fold compared with LNCaP/GFP cells.
Since CAMKK2 overexpression down-regulated
PSA promoter activity in the presence and absence of
DHT, we also explored the effect of CAMKK2 overexpression on LNCaP cell proliferation. The proliferation rate of LNCaP/GFP-CAMKK2 cells without
DHT was diminished compared with LNCaP/GFP
cells (P < 0.05; Fig. 3B). Moreover, whereas theFig. 3. Effect of CAMK2 overexpression on LNCaP cells. A: After transfection of LNCaP/GFP and LNCaP/GFP-CAMKK2 cells with
pGL3PSAp-5.8, cells were treated with 0, 0.1, or1.0 nMDHT for 24 hr and luciferase activity was examined.B: After LNCaP/GFP and LNCaP/
GFP-CAMKK2 cells were treated with 0, 0.1, 1.0, and 10 nM DHT for 4 days, the numbers of LNCaP/GFP and LNCaP/GFP-CAMKK2 cells
were counted. C: After 2  106 LNCaP/GFP, LNCaP/GFP-CAMKK2 cells were injected into dorsal subcutis of intact SCID mice, thevolume of dorsal tumor was monitored every 3 or 4 days. All values are expressed as mean  SE. D: Enzyme immunoassay of PSA was
performed in serum of SCID mice that were sacrificed. E: After 2  106 LNCaP/GFP, LNCaP/GFP-CAMKK2 cells were injected into dorsal
subcutis of castrated SCID mice, thevolume of dorsal tumor wasmonitored.F: Enzymeimmunoassayof PSAwas also performed as (D).
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proliferation of LNCaP/GFP cells was stimulated
in 0.1 nM DHT most, the proliferation of LNCaP/
GFP-CAMKK2 cells was stimulated in 1 nM DHT
most (P < 0.001; Fig. 3B). This result suggested thatthe androgen-sensitivity of LNCaP cells was diminished by CAMKK2 overexpression.
To assess the effect of CAMKK2 overexpression on
tumorigenesis in xenograft model, LNCaP/GFP and
LNCaP/GFP-CAMKK2 cells were injected subcutaneously in dorsal flank of intact male SCID mice. The
growth rate of LNCaP/GFP-CAMKK2 cells was significantly declined to 25% of LNCaP/GFP cells at
day 115 (P ¼ 0.017; Fig. 3C). In accordance withgrowth, mean serum PSA value on sacrifice in
LNCaP/GFP-CAMKK2-bearing mice was decreased
by 4.2-fold compared with LNCaP/GFP-bearing mice
(P < 0.01; Fig. 3D). We simultaneously assessed
the tumorigenesis of LNCaP/GFP-CAMKK2 cells
in male SCID mice castrated 1 week ago. Eventually,
the growth rate of LNCaP/GFP and LNCaP/GFPCAMKK2 cells was decrease in castrated mice compared with intact mice (compare Fig. 3C and E). Thenthe growth rate of LNCaP/GFP-CAMKK2 cells was
declined to 15% of LNCaP/GFP cells in castrated
mice at day 130 (P < 0.05; Fig. 3E). Serum PSA value
on sacrifice in LNCaP/GFP-CAMKK2-bearing micealso decreased by 8.8-fold compared with LNCaP/
GFP-bearing mice (P < 0.05; Fig. 3F). These results
suggested that CAMKK2 overexpression in LNCaP
cells inhibit cell proliferation in vitro and in vivo irrespective of androgen.
Knockdown of CAMKK2 Induces AR Activity
Since significant overexpression of a certain proteinusing a strong artificial promoter, such as CMV promoter, might cause a stress on the cell and thus decrease its growth rates. We, therefore, reduced
CAMKK2 expression using small interfering RNA
conversely. Two different siRNA (siCAMKK2-01 and02) were first employed for knockdown of CAMKK2.
Knockdown of CAMKK2 by siCAMKK2-01 and 02 repressed CAMKK2 mRNA expression and CAMKK2
protein expression by order of CAMKK2-01 and 02(Fig. 4A). We evaluated the effect of knockdown of
CAMKK2 on PSA promoter activity in LNCaP cells
by luciferase reporter assay. In accordance withknockdown of CAMKK2, PSA promoter activity was
inversely correlated with CAMKK2 protein expression (Fig. 4B). Especially, knockdown of CAMKK2 by
20 nM siCAMKK2-01 enhanced 3.6 times PSA promoter activity induced by 0.1 nM DHT comparedwith control non-target siRNA (siNT). Therefore, wechose siCAMKK2-01 for further knockdown study of
CAMKK2. Knockdown of CAMKK2 by siCAMKK2The Prostateenhanced PSA promoter activity induced by 0.1 nM
DHT from 1.9 to 4.1 times compared with siNT in adose-dependent manner (Fig. 4C). We further investigated if knockdown of CAMKK2 expression affectdifferent androgen-inducible MMTV promoter activity. MMTV promoter induced by 1.0 nM DHT aswell as PSA promoter was enhanced by knockdown
of CAMKK2 (Fig. 4D). In accordance with inductionof PSA promoter activity by knockdown of CAMKK2,LNCaP cell proliferation was stimulated by knockdown of CAMKK2 (Fig. 4E). Especially, proliferation
stimulation by the addition of 0.1 nM DHT was
further enhanced by knockdown of CAMKK2, suggesting that androgen-sensitivity was enhanced bydown-regulation of CAMKK2 expression.
In order to confirm whether a phenomenon observed with LNCaP cells applied to other cells, weused androgen-sensitive PCa VCaP cells and performed a similar experiment. DHT-induced PSA and
MMTV promoter activity in VCaP cells was enhancedby knockdown of CAMKK2, suggesting that this phenomenon is common events observed in androgensensitive cells (Fig. 4F).
Since overexpression of CAMKK2-reduced AR
protein level in the presence of DHT as describedabove, we explored AR expression level on knockdown of CAMKK2 by siCAMKK2. As we expected,
AR expression level that was increased 24 hr after
DHT-treatment was further elevated by knockdown
of CAMKK2, although it was not changed in the absence of DHT by knockdown of CAMKK2 (Fig. 5A).
To further confirm if basal PSA promoter activity induced by knockdown of CAMKK2 is related with
AR activity, we employed another reporter vector
driven by a 633 bp deleted PSA promoter that
includes only two weak AREs (pGL3PSAp-0.63) for
this experiment. As compared with pGL3PSAp-5.8
that includes strong ARE3, LNCaP cells transfectedwith pGL3PSAp-0.63 showed weak induction of PSA
promoter activity by DHT (two times). Interestingly,
not only basal PSA promoter activity but also DHTinduced PSA promoter activity of pGL3PSAp-0.63
was not enhanced by knockdown of CAMKK2(Fig. 5B). These results suggested that enhancement of
AR promoter activity by knockdown of CAMKK2
might be mediated by not only AR stability but also
other transcriptional factors related with ARE3.
DISCUSSION
In the present study, we demonstrated thatCAMKK2 identified by cDNA microarray technique
had a function as a putative tumor suppressor andthat down-regulation of CAMKK2 causes androgenhypersensitivity during ADT.

Repression of CAMKK2 Induces Androgen-Hypersensitivity

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Fig. 4. Effect of knockdown of CAMKK2 expression on AR activity. A: Knockdown of CAMKK2 expression in LNCaP cells by CAMKK2
siRNA- 01 and - 02 transfection.Twenty-four hours or 72 hr after transfection with NTsiRNA or CAMKK2 siRNA- 01 or - 02, total RNA or
total proteins from LNCaP cells were extracted and subjected to RT-PCR or Western blot analysis of CAMKK2 and GAPDH, respectively.
B: Enhancement of PSA promoter activity by knockdown of CAMKK2. PSA promoter activity induced by DHTwas examined after transfection with NT siRNA or CAMKK2 siRNA- 01 and - 02. C: After LNCaP cells were transfected with 5, 10, or 20 nM NT siRNA or CAMKK2
siRNA for 12 hr, cells were transfected with pGL3PSAp-5.8. D: After LNCaP cells were transfected with NT siRNA or CAMKK2 siRNA,cells were transfected with pGL3PSAp-5.8 (PSA promoter) or pGL3MMTV (MMTV promoter and were treated with 0.1 nM DHTor1.0 nM
DHT for 24 hr, respectively. E: Effect of knockdown of CAMKK2 expression on LNCaP cell proliferation. F: Effect of knockdown of
CAMKK2 on PSA promoter activityinVCaPcells.Knockdown o CAMKK2 andluciferase assay wereperformed as describedin (D).

Eventually CAMKK2 protein was expressed morein PCa tissue samples than in normal prostate
tissue in accordance with cDNA microarray analysis
between normal prostate and PCa samples,suggesting that CAMMKK2 can become the usefulimmunohistochemical biomarker of PCa. However,
the CAMKK2-negative staining in tissue microarraysamples was rather increased in PCa patients
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Fig. 5. Effectof knockdown of CAMKK2 on AR activityin LNCaPcells. A: After LNCaPcells were transfected with NTsiRNA or CAMKK2
siRNA, cells were treated with 0 or10 nM DHT for 24 hr and whole cell lysates were extracted for Western blot analysis of AR and GAPDH.
B: LNCaP cells were transfected with NT siRNA or CAMKK2 siRNA. Then LNCaP cells were transfected with pGL3PSAp- 0.63 or
pGL3PSAp-5.8 and treated with 0 or1 nMDHT for 24 hr, andluciferase assay was performed.

with high Gleason score. Moreover, when an expression of CAMKK2-positive area was narrow in PCa tissue, PSA-progression free survival rate after first
ADT was low. The phenomenon that CAMKK2 isexpressed in PCa more than in normal prostate may
be contribution to the carcinogenesis of PCa. However, once PCa is formed to some size, a function of
CAMKK2 may changes and CAMKK2 may act tomake a cell proliferation inhibited. In contrast,
among the various cells and organs, homeostasis isoften preserved by the feedback function through various hormones and cytokines. Even if it is PCa cells,
expression of CAMKK2 that is promoted by androgenmay regulate a rapid cell proliferation by negative
feedback, so called forming a negative regulatory
loop to maintain a balance in proliferation between
androgen and CAMKK2. It is well-known phenomenon that the proliferation of LNCaP cells shows biphasic pattern on androgen concentration: DHT of
the high concentration rather inhibited the LNCaP
proliferation [26] (Figs. 3B and 4E). Nevertheless,
CAMKK2 expression was induced by DHT in a dosedependent manner (Fig. 2A). Furthermore, overexpression of CAMKK2 repressed the proliferation of
LNCaP cells and attenuated androgen-sensitivity,
and that down-regulation of CAMKK2 stimulated cellproliferation and enhanced androgen-sensitivity for
DHT of the low concentration. Thus, low expressionof CAMKK2 as a tumor suppressor before ADT or
down-regulation of CAMKK2 by ADT may contribute
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relapse of PCa in response to adrenal androgen of the
low concentration at later stage. This similar phenomenon is also observed in suppressors of cytokine
signaling (SOCS)-3. SOCS-3 as well as CAMKK2 are
up-regulated by androgen in PCa cell lines and inhibit
androgen-mediated proliferation [27].
How does CAMKK2 expression repress AR-signaling axis and proliferation? One of its mechanisms
may be involvement of AR protein. Overexpression of
CAMKK2 repressed AR protein level (Fig. 2). Pelley
et al. described that Ca2þ stimulated AR breakdownin vitro and in vivo and that calmodulin bound to AR
seemed to play a role in calpain-mediated cleavageof AR in LNCaP cells [28,29]. Together, the effect of
CAMKK2 on AR protein stability in the presence of
androgen may change AR-signaling axis.
The other reason for influence of CAMKK2 on cellproliferation may be the activation of AMPK by
AMPK phosphorylation. CAMKK2 is upstream protein kinase for AMPK [30]. Zhou et al. demonstrated
that inactivation of AMPK by dominant negative
AMPK mutant accelerated cell proliferation and promoted malignant behavior such as increased
cell migration and anchorage-independent proliferation mediating through lipopolysaccharide-induced
tumor necrosis factor (TNF) alpha factor (LITAF) andTNF superfamily member 15 (TNFSF15) [31,32].
Serine-threonine liver kinase B1 (LKB1) known as atumor suppressor also activates AMPK, and have crucial roles in cell proliferation [33]. One of the majorRepression of CAMKK2 Induces Androgen-Hypersensitivity

1799

Fig. 6. Function of CAMKK2 on PCa.Therole of CAMKK2 on PCa growth and a mechanism of transition to CRPC during ADT.

proliferation regulatory pathways controlled by
LKB1–AMPK is the mTOR (mammalian Target ofRapamycin) pathway. The mTOR pathway is inhibited through tuberous sclerosis complex 2 (TSC2)
known as a tumor suppressor [33]. Activation of
AMPK by CAMKK2 overexpression might repress
PCa proliferation through mTOR inhibition as wellas LKB1. Park et al. [34] demonstrated that AMPK
was highly activated in 40% of human PCa and act asa metabolic survival factor. In breast cancer, reduced
phsphorylated AMPK signal was significantly associated with higher histological grade and axillary nodemetastasis [35]. These reports suggest that the regulation of CAMKK2 on cell proliferation in PCa is mediated through AMPK activation.
However, the function of CAMKK2-AMPK pathway is still controversial [19,20]. Frigo et al. [19] andMassie et al. [20] demonstrated that CAMKK2induced migration and invasion of LNCaP cells andstimulated LNCaP cell growth mediating throughphosphorylation of AMPK. IHC in our study revealed
that the ratio of CAMKK2-negative staining in the
PCa with the high-grade Gleason score that has

more malignant potential was increased comparedwith the low-grade Gleason score. Furthermore, ourpresent study that CAMKK2-AMPK pathway inhibited PCa growth is rather coincident with Zhou et al.
[31] and Hadad et al. [35]. It remains unclear why
CAMKK2-AMPK pathway show different functions.
The function of CAMKK2 may change at different situation, such as late stage of CRPC. Further study will
be needed to clarify this pathway.
As described in Figure 6, these observations raise
the intriguing mechanism that hormone-naive PCathat initially responds to ADT becomes castrationresistant. Before hormonal therapy, CAMKK2 isexpressing in response to androgen. It may be thecause why a proliferation rate of the PCa is generally
slow compared with other cancers, such as lungcancer and colon cancer that expression of
CAMKK2 controls excessive growth of PCa throughactivation of AMPK or other signals. However, when
ADT is conducted for PCa, expression of CAMKK2 inthe PCa attenuates, and activation of AMPK is repressed. As a result, PCa changes in the state that isandrogen-hypersensitive, and androgen of the low
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1800

Shima et al.

concentration promotes growth of the PCa and shifts
in CRPC.
Atorvastatin known as a statin famous for the control of hypercholesterolemia also activates CaMKK2
as an upstream kinase of AMPK in endothelium [36].
Recently, Zheng et al. demonstrated that administration of a combination of atorvastatin and celecoxib
might be an effective strategy for the prevention of
PCa LNCaP cell progression from androgen dependence to androgen independence in castrated SCID
mice [37]. Although down-regulation of CAMKK2
assists recurrence as described above, the continuous
activation of CAMKK2 with some reagents such as
atorvastatin may prevent recurrence during ADT.
In conclusion, we suggest that ADT downregulates CAMKK2 and that CAMKK2 subsequently
stabilize AR that is a key mediator of hormonal signaling. Moreover, the finding that CAMKK2 expression is reduced in more malignant PCa suggests thatloss of CAMKK2 may enhance androgen signaling
and promote prostate cell proliferation. Furthermore,
the fact that overexpression of CAMKK2 in vivo
reduces tumor growth suggests that reactivation of
CAMKK2 by development of new drugs may be a
new strategy to prolong the period to respond ADT
and inhibit PCa progression.
ACKNOWLEDGMENTS
We thank Y. Kawabuchi for skilled technicalassistance (Kanazawa University). This study was
supported by Grant-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education,
Culture, Sport, Science, and Technology of Japan(21592037).
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